A novel series of thiazolyl-pyrazoline derivatives: Synthesis and evaluation of antifungal activity, cytotoxicity and genotoxicity

Article abstract of DOI:10.1016/j.ejmech.2014.12.055

In the current work, new thiazolylepyrazoline derivatives (1e22) were synthesized and evaluated for their antifungal effects against pathogenic yeasts and molds using a broth microdilution assay. Ames assay was carried out to determine the genotoxicity of

Full text of DOI:10.1016/j.ejmech.2014.12.055

European Journal of Medicinal Chemistry 92 (2015) 342e352
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel series of thiazolylepyrazoline derivatives: Synthesis and
evaluation of antifungal activity, cytotoxicity and genotoxicity
,
*
a
a
b
Mehlika Dilek Altıntop a , Ahmet Ozdemir , Gülhan Turan-Zitouni , Sinem Ilgın ,
€
b
c
a
€
Ozlem Atlı , Rasime Demirel , Zafer Asım Kaplancıklı
a Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskis¸ehir, Turkey
b Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 26470 Eskis¸ehir, Turkey
c Anadolu University, Faculty of Science, Department of Biology, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In the current work, new thiazolylepyrazoline derivatives (1e22) were synthesized and evaluated for
their antifungal effects against pathogenic yeasts and molds using a broth microdilution assay. Ames
assay was carried out to determine the genotoxicity of the most effective antifungal derivatives. The
cytotoxicity of the compounds (1e22) was also investigated against A549 human lung adenocarcinoma
and NIH/3T3 mouse embryonic fibroblast cells. Among these derivatives, 2-[5-(4-fluorophenyl)-3-(5-
methylthiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]-4-(4-methylsulfonylphenyl)thiazole (18) can be
identified as the most promising anticandidal derivative due to its notable inhibitory effect on Candida
Received 7 August 2014
Received in revised form
6 November 2014
Accepted 30 December 2014
Available online 31 December 2014
Keywords:
Thiazole
Pyrazoline
Antifungal activity
Cytotoxicity
Genotoxicity
zeylanoides with a MIC value of 250
m
g/mL when compared with ketoconazole (MIC ¼ 250
mg/mL), low
cytotoxicity against NIH/3T3 cells and non-mutagenic effect. On the other hand, 2-[5-(4-fluorophenyl)-3-
(5-chlorothiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]-4-(4-bromophenyl)thiazole (4) can be considered
as the most promising anticancer agent against A549 cancer cells owing to its notable inhibitory effect on
A549 cells with an IC50 value of 62.5
cytotoxicity against NIH/3T3 cells.
m
g/mL when compared with cisplatin (IC50 ¼ 45.88
mg/mL) and low
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
class of ‘antimicrobial agents’ [9e11]. Furthermore, a considerable
amount of research has reported that pyrazole-based heterocycles
show promising activity against cancer cell lines including A549
human lung adenocarcinoma cell lines [12e16].
Eukaryotic pathogens such as fungi pose a continuous and
serious threat to public health since they share a close evolutionary
relationship with their human hosts, limiting the number of drug
targets that can be exploited to selectively kill fungal pathogens [1].
In recent years, the acquisition of multiple-drug resistance has
resulted in a corresponding increase in demand for new effective
antifungal agents with enhanced activity and limited toxicity [2e4].
On the other hand, in the last few decades, cancer has emerged as
the second leading cause of death after cardiovascular disorders.
The treatment of cancer is often complicated by high toxicity, low
tolerability, and development of resistance [5e8].
In terms of medicinal chemistry, thiazoles have also attracted a
great deal of interest due to their presence in a large number of
biologically active compounds, including natural products and
pharmaceutical agents. The clinical efficacy of tiazofurin and its
analogs, and bleomycins (BLMs) pointed out the importance of
thiazole ring in the field of cancer treatment. Sulfathiazole (anti-
microbial drug), abafungin (antifungal drug), and ritonavir (anti-
viral drug) are other examples of thiazole-based agents.
Considerable research on thiazole and thiazolylepyrazoline de-
rivatives in relation to their biological activity has been accom-
plished [17e32].
Prompted by the afore-mentioned findings and in the continu-
ation of our ongoing research in the field of design, synthesis and
biological evaluation of thiazolylepyrazoline derivatives [33,34],
herein we described the synthesis and evaluation of a new series of
thiophene substituted thiazolylepyrazolines as potential
Medicinal chemists have carried out considerable research on
pyrazoline derivatives due to their diverse therapeutic applications
extending from central nervous system applications to antimicro-
bials. The most predominant biological activity is observed for the
* Corresponding author.
E-mail address: mdaltintop@anadolu.edu.tr (M.D. Altıntop).
http://dx.doi.org/10.1016/j.ejmech.2014.12.055
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
343
antifungal and anticancer agents.
2. Results and discussion
The synthesis of thiazolylepyrazoline derivatives (1e22) fol-
lowed the general pathway outlined in Scheme 1. Initially, 1-(5-
chloro/methylthiophen-2-yl)-3-(4-fluorophenyl)-2-propen-1-ones
(A/B) were synthesized via the base-catalyzed ClaiseneSchmidt
condensation of 2-acetyl-5-chloro/methylthiophene with 4-
fluorobenzaldehyde. Secondly, 3-(5-chloro/methylthiophen-2-yl)-
5-(4-fluorophenyl)-1-thiocarbamoyl-2-pyrazolines (C/D) were ob-
tained by the cyclization of chalcones (A/B) with thiosemicarbazide
in the presence of sodium hydroxide. Finally, the ring closure re-
action of 3-(5-chloro/methylthiophen-2-yl)-5-(4-fluorophenyl)-1-
thiocarbamoyl-2-pyrazolines (C/D) with phenacyl bromides affor-
ded thiazolylepyrazoline derivatives (1e22).
The structures of the newly synthesized compounds were
elucidated by IR, 1H NMR, 13C NMR, mass spectral data, and
elemental analyses.
Fig. 1. AMX system of the pyrazoline ring.
(Fig. 1). All the other aromatic and aliphatic protons were observed
at expected regions.
In the IR spectra of compounds C and D, the stretching bands for
NeH group were observed in the region 3473e3350 cmꢀ1. C]N,
C]C stretching and NeH bending vibrations were observed in the
region 1575e1454 cmꢀ1. In the IR spectra of compounds 1e22, C]
N, C]C stretching and NeH bending vibrations were observed in
the region 1633e1450 cmꢀ1. The aromatic and aliphatic CeH
stretching vibrations gave rise to bands at 3140e3016 cmꢀ1 and
2987e2839 cmꢀ1, respectively. In the IR spectra of the cyano-
substituted compounds, the stretching bands for C^N group
In the 13C NMR spectra of the compounds, the C3, C4 and C5
carbons of the pyrazoline ring were observed at 148e151 ppm,
42e44 ppm and 62e65 ppm, respectively. In the 13C NMR spectra
of compounds C and D, C]S carbon appeared in the region
175e176 ppm. All the other aromatic and aliphatic carbons were
observed at expected regions.
All of the synthetic compounds gave satisfactory mass spec-
troscopic data and elemental analysis, which were in full accor-
dance with their depicted structures.
The synthesized compounds (1e22) were tested in vitro against
pathogenic yeasts and molds. As shown in Table 1, the compounds
exhibited more significant antifungal activity against yeasts than
molds.
occurred at 2223e2218 cmꢀ1
.
In the 1H NMR spectra of the compounds, the CH2 protons of the
pyrazoline ring resonated as a pair of doublets of doublets at
d
3.14e3.44 ppm (HA), 3.98e4.04 ppm (HM). The CH proton
appeared as doublet of doublets at 5.64e5.72 (HX) ppm due to
vicinal coupling with two magnetically non-equivalent protons of
the methylene group at position of the pyrazoline ring
(JAM ¼ 17.50e18.00 Hz, JAX ¼ 6.50e7.50 Hz, JMX ¼ 11.50e12.00 Hz)
d
Among the pathogenic fungi species, Candida zeylanoides was
the most susceptible yeast to the tested compounds. Compounds
4
Scheme 1. The synthetic route for the preparation of thiazolylepyrazoline derivatives (1e22). Reagents and conditions: (i) 10% aqueous sodium hydroxide solution, ethanol, rt, 10 h;
(ii) thiosemicarbazide, NaOH, ethanol, reflux, 8 h; (iii) substituted 2-bromoacetophenone, ethanol, reflux, 6 h.

344
M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
Table 1
Antifungal effects of the compounds (1e22) as MIC values (mg/mL).
Compound
A
B
C
D
E
F
G
H
I
J
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
125
250
125
125
125
125
250
125
125
125
125
125
125
125
125
125
125
125
250
250
250
250
250
250
250
250
250
250
250
250
250
250
500
500
250
250
250
250
250
250
500
500
250
250
250
250
250
250
250
250
250
250
250
250
500
500
250
250
250
250
250
250
500
500
250
250
125
250
250
250
250
250
250
250
250
250
250
250
500
500
250
250
250
250
250
250
500
500
250
250
250
250
250
250
250
250
250
250
250
250
250
500
500
250
250
250
250
250
250
500
500
250
250
125
125
125
125
250
250
250
250
250
250
125
250
250
250
250
250
125
125
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
1000
500
250
250
250
250
250
250
500
500
250
250
125
17
18
19
20
21
22
Ketoconazole
62.5
62.5
15.62
15.62
31.25
7.81
62.5
A: C. glabrata (Clinical Isolate, Osmangazi University, Faculty of Medicine, Eskis¸ ehir, Turkey), B: C. albicans (ATCC-90028), C: C. tropicalis (NRLL Y-12968), D: C. krusei (NRLL Y-
7179), E: C. zeylanoides (NRLL Y-1774), F: C. parapsilosis (NRLL Y-12696), G: A. flavus (NRRL-980), H: A. niger (ATCC-1094), I: A. parasiticus (NRRL-465), J: F. solani (NRRL-13414).
11, 12, 19 and 20 exhibited their inhibitory effects on C. zeylanoides
with a MIC value of 500
ketoconazole showed their anticandidal activity with a MIC value of
250 g/mL. Compounds 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
and 18 showed their antifungal activity against Candida glabrata
with a MIC value of 125 g/mL, whilst ketoconazole displayed its
anticandidal activity with a MIC value of 62.5 g/mL. Compounds 1,
mg/mL, whereas other derivatives and
Table 2
AMESMPF results of the compounds (1e22).
m
Compound
Concentrations (mg/mL)
Fold increase over the baseline
TA 98
TA 100
m
S9þ
S9ꢀ
S9þ
S9ꢀ
m
2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 18, 21 and 22 showed their
inhibitory effects on Candida tropicalis and Candida krusei with a
1
0.156
0.3125
0.625
1.25
2.5
1.63*
0.68
1.08
0.68
0.27
0.41
0.68
0.81
0.95
0.81
0.54
0.81
0.68
0.81
0.95
0.81
0.68
0.81
0.87
0.55
1.09
0.22
0.33
0.98
0.27
0.18*
1.07*
0.44
0.89
1.16
0.41
0.27
1.63*
0.95
1.36*
0.81
0.00
0.00
0.67
0.00
0.00
0.67
0.00
0.00
0.00
0.67
0.67
1.00
0.00
0.32
0.00
0.00
0.00
0.00
0.00
0.33
0.67
0.67
0.00
0.67
1.78*
1.56*
1.11*
1.11
1.56*
1.33*
0.67
0.22
0.44
0.89
0.67
0.89
1.85*
3.23**
2.77**
4.62**
3.46**
4.38**
3.23**
3.92**
3.46**
3.69**
3.00**
3.92**
2.77**
3.92**
3.92**
5.08**
3.92**
2.54**
2.08**
2.31**
2.54**
2.77**
2.31**
2.31**
1.01
0.00
0.32
0.96
1.28
2.88**
0.96
1.67*
0.67
0.33
0.33
1.00*
0.33
1.00
1.00
0.33
0.00
0.33
2.00**
0.67
1.00
1.33
3.00**
1.33
2.67*
0.80
0.74
0.74
0.31
0.31
0.80
0.78
1.04*
0.99
0.52
0.52
1.09*
MIC value of 250
(MIC ¼ 125 g/mL). On the other hand, all derivatives showed their
antifungal activity against Aspergillus niger and Fusarium solani
with a MIC value of 250 g/mL, whilst ketoconazole exhibited its
inhibitory effects on A. niger and F. solani with MIC values of
31.25 g/mL and 62.5 g/mL, respectively. The microbiological re-
mg/mL when compared with ketoconazole
m
5
m
3
0.156
0.3125
0.625
1.25
2.5
m
m
sults demonstrated that the antifungal effects of these compounds
on A. niger and F. solani did not depend on the substituents. Com-
pounds 1, 2, 3, 4, 11, 17, and 18 exhibited their antifungal activity
5
4
0.156
0.3125
0.625
1.25
2.5
against Aspergillus flavus with a MIC value of 125
ketoconazole showed its antifungal activity with a MIC value of
15.62 g/mL. Considering the antifungal effects of the compounds
mg/mL, whereas
m
against pathogenic yeasts and molds, compounds 1, 3, 4, 11, 17 and
18 were selected to undergo further studies.
5
11
17
18
0.156
0.3125
0.625
1.25
2.5
Ames assay was carried out to evaluate the genotoxicity of the
most effective antifungal derivatives. In Ames MPF assay, more than
25 positive wells were observed with positive controls. This com-
plied with the requirements in the Ames MPF assay manual.
Negative controls showed less than 8 positive wells in the presence
and absence of S9 with TA98 and TA100. These complied with the
requirements in the Ames MPF assay manual and previous studies
[35]. Our results were presented in Table 2.
Compounds 1, 3 and 4 had a baseline of 2.46 with TA 98 in the
presence of S9 and 1.0 in the absence of S9 with related negative
control values. Furthermore, fold induction over baseline was less
than 2 in each concentration of the compounds and the significant
different results obtained did not show a dose-response tendency.
So, the results of the Ames test indicated the nonmutagenic po-
tentials of compounds 1, 3 and 4 against TA 98. Compounds 1, 3 and
4 showed a baseline of 1.44 and 1.04 against TA100 with/without
5
0.156
0.3125
0.625
1.25
2.5
0.94
0.86
0.70
0.55
1.01
0.73
0.73
0.93
5
0.156
0.3125
0.625
1.25
2.5
0.80
1.26*
0.93
5
* t test p value (unpaired 1-sided) < 0.05; ** t test p value (unpaired 1-sided) < 0.05
with a fold-induction over baseline > 2.

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
345
Table 3
S9, respectively. Fold inductions over baseline were also more than
IC50 values of the compounds (1e22) against A549 and NIH/3T3 cell lines (
m
g/mL).
3 and statistically different results were obtained in the presence of
S9 against TA 100. Partially, compound 4 showed 2-fold increase
over baseline, which was also statistically significant, in its highest
concentration without S9 against TA 100. According to these re-
sults, compounds 1, 3 and 4 showed mutagenic properties against
TA 100 in the presence of S9. Furthermore, compound 4 can be
considered as a weak mutagen against TA 100 without S9.
Compound 11 had a baseline of 3.06 against TA 98 in the pres-
ence of S9 and 1.0 in the absence of S9. Fold inductions over
baseline were also less than 2 in each concentration of the com-
pounds and the significant different results obtained did not show a
dose-response tendency. So, the Ames test indicated the non-
mutagenic potential of compound 11 against TA 98. The baseline
values obtained was 1.44 and 1.04 against TA100 in the presence
and absence of S9, respectively. This compound also showed 2e3
fold increases over the baseline which were statistically significant
and showed a dose-responce tendency. According to the criteria
obtained from previous studies, compound 11 can be classified as
weak positive against TA 100 with/without S9 (Fig. 2).
Mentioned-fold increases over the baseline according to the
criteria were not determined with compounds 17 and 18. Therefore,
compounds 17 and 18 were classified as non-mutagenic com-
pounds against TA 98 and 100 with/without S9. Although com-
pounds 17 and 18 are 5-methylthiophene substituted
thiazolylepyrazoline derivatives, methylsulfonyl-substituted com-
pound 18 is less cytotoxic than cyano-substituted compound 17
against NIH/3T3 mouse embryonic fibroblast cells. It can be
concluded that cyano substituent increased cytotoxicity.
Compound
IC50 (mg/mL)
A549 cell line
NIH/3T3 cell line
1
2
3
4
5
6
7
8
>500
>500
125
>500
>500
>500
>500
>500
>500
>500
>500
102.96
73.77
>500
>500
>500
>500
>500
15.6
62.5
>500
>500
>500
>500
>500
>500
>500
>500
125
>500
>500
16.67
321.8
>500
>500
183.65
173.44
>500
45.88
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Cisplatin
250
373.83
>500
146.5
125
>500
ND
ND: Not Determined.
A549 cell line than the other derivatives. On the other hand, com-
pounds 9, 10, 16, 17, 20 and 21 exhibited more cytotoxicity against
NIH/3T3 cell line than the other compounds.
In terms of their anticancer potential, compounds 3, 4 and 13
can be considered to be selective cytotoxic agents against A549 cell
line due to their low cytotoxicity against NIH/3T3 cell line.
According to the genotoxicity and cytotoxicity assays, com-
pound 18 seems to be the most promising anticandidal derivative
due to its notable inhibitory effect on C. zeylanoides with a MIC
value of 250 mg/mL, low cytotoxicity against NIH/3T3 cells and non-
mutagenic effect.
The compounds were investigated for their antiproliferative
effects on A549 human lung adenocarcinoma cell line. Additionally,
these compounds were tested for their cytotoxicity against NIH/3T3
mouse embryonic fibroblast cell line (Table 3). Compounds 3, 4, 13,
16, 17, 20 and 21 showed higher antiproliferative activity against
Among these compounds, compound
chlorothiophene and p-bromophenyl moieties showed the high-
est cytotoxicity against A549 cell line with an IC50 value of 62.5 g/
mL, which was very similar to the positive control cisplatin
(IC50 ¼ 45.88 g/mL). Although compound 15 also carries p-bromo
substituent on phenyl ring, compound 15 showed low cytotoxicity
against A549 cell line (IC50 > 500 g/mL). This result pointed out
4
bearing 5-
m
m
m
Fig. 2. Dose-response curve of compound 11 against TA98 and TA100 in the presence and absence of S9 according to AMES MPF test. * :t test p value (unpaired 1-sided) < 0.05 with a
fold-induction over baseline > 2; : 2-fold induction over baseline.

346
M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
the importance of 5-chlorothiophene moiety for anticancer activity
against A549 cell line.
According to our studies performed, compound 4 showed
promising cytotoxic activity against A549 cell line with a similar
dose of cisplatin without causing any toxicity on NIH/3T3 mouse
embryonic fibroblast cell line.
crystallized from ethanol.
4.1.1.2.1. 3-(5-Chlorothiophen-2-yl)-5-(4-fluorophenyl)-1-
thiocarbamoyl-2-pyrazoline (C). Yield: 85%. Mp 230e231 ꢁC.
IR nmax (cmꢀ1): 3473.80, 3350.35 (NeH stretching), 1575.84,
1508.33, 1475.54 (C]N and C]C stretching), 1438.90, 1415.75,
1365.60, 1338.60 (CeH bending), 1215.15, 1163.08, 1091.71, 1012.63
(CeN stretching and aromatic CeH in plane bending), 835.18,
794.67 (aromatic CeH out of plane bending).
3. Conclusion
1H NMR (400 MHz, DMSO-d6)
d (ppm): 3.14 (1H, dd,
In this study, new thiazolylepyrazoline derivatives were syn-
thesized and investigated for their antifungal activity and cyto-
toxicity against A549 cell line.
JAM ¼ 18.0 Hz, JAX ¼ 3.6 Hz, C4-HA pyrazoline), 3.88 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 11.6 Hz, C4-HM pyrazoline), 5.95 (1H, dd,
JMX ¼ 11.2 Hz, JAX ¼ 3.6 Hz, C5-HX pyrazoline), 7.15e7.37 (6H, m,
aromatic protons), 7.68 (1H, brs, NeH), 8.11 (1H, brs, NeH).
An ideal drug is expected to exhibit high therapeutic effect and
minimum toxicity. During early drug testing, Ames assay is widely
used by the pharmaceutical industry to assess the mutagenic po-
tential of the drug candidates for their reliability. For this purpose,
Ames test was performed to determine the genotoxicity of the most
effective antifungal compounds in the present study. Furthermore,
a potential agent should have minimum or no side-effects on
normal cells. So, in the current study the cytotoxic effects of all
compounds were also investigated on NIH/3T3 cell lines.
In particular, compound 18 was the most promising antifungal
13C NMR (100 MHz, DMSO-d6)
d (ppm): 42.34 (CH2), 62.41 (CH),
115.07 and 115.29 (2CH), 127.31 (CH), 127.39 (CH), 127.94 (C), 130.83
(C), 132.10 (CH), 132.81 (CH), 138.68 and 138.71 (C), 150.11 (C),
159.90 and 162.32 (C), 175.89 (C).
For C14H11ClFN3S2 Calculated: C, 49.48; H, 3.26; N, 12.36. Found:
C, 49.49; H, 3.24; N, 12.35.
MS (ESI) (m/z): (Mþþ1) 340.8.
4.1.1.2.2. 3-(5-Methylthiophen-2-yl)-5-(4-fluorophenyl)-1-
thiocarbamoyl-2-pyrazoline (D). Yield: 78%. Mp 225e226 ꢁC.
IR nmax (cmꢀ1): 3473.80, 3352.28 (NeH stretching), 3051.39
(aromatic CeH), 2916.37 (aliphatic CeH), 1575.84, 1506.41, 1489.05,
1454.33 (C]N and C]C), 1361.74, 1342.46 (CeH bending), 1213.23,
1163.08, 1091.71 (CeN stretching and aromatic CeH in plane
bending), 835.18, 798.53 (aromatic CeH out of plane bending).
derivative against C. zeylanoides with a MIC value of 250
mg/mL
when compared with ketoconazole. In addition, this agent did not
show any genotoxic potential and the cytotoxic dose of the com-
pound was higher than its effective dose. Furthermore, compound
4 can be identified as the most promising anticancer agent owing to
its antiproliferative effect on A549 cancer cell lines and non-toxic
potential against NIH/3T3 cells. Further studies are required to
evaluate the mechanism of action for the anticancer activity of
compound 4 and anticandidal activity of compound 18.
1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.48 (3H, s, CH3), 3.10
(1H, dd, JAM ¼ 17.6 Hz, JAX ¼ 3.2 Hz, C4-HA pyrazoline), 3.87 (1H, dd,
JMA ¼ 17.6 Hz, JMX ¼ 11.6 Hz, C4-HM pyrazoline), 5.93 (1H, dd,
JMX ¼ 11.6 Hz, JAX ¼ 3.2 Hz, C5-HX pyrazoline), 6.85 (1H, dd, J ¼ 3.6,
1.2 Hz, thiophene C4eH), 7.14e7.29 (5H, m, aromatic protons), 7.52
(1H, brs, NeH), 8.04 (1H, brs, NeH).
4. Experimental
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.21 (CH3), 42.77 (CH2),
4.1. Chemistry
62.13 (CH), 115.05 and 115.26 (2CH), 126.56 (C), 127.27 (CH), 127.36
(CH), 131.30 (CH), 131.43 (CH), 138.80 and 138.83 (C), 144.41 (C),
151.06 (C), 159.88 and 162.29 (C), 175.57 (C).
All reagents were purchased from commercial suppliers and
used without further purification. Melting points (Mp) were
determined on an Electrothermal 9100 melting point apparatus
(Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR
spectra were recorded on an IRPrestige-21 Fourier Transform
Infrared spectrophotometer (Shimadzu, Tokyo, Japan). 1H NMR and
13C NMR spectra of the synthesized compounds were recorded on a
Bruker spectrometer (Bruker, Billerica, USA). Mass spectra were
recorded on an Agilent LC-MSD-Trap-SL Mass spectrometer (Agi-
lent, Minnesota, USA). Elemental analyses were performed on a
Perkin Elmer EAL 240 elemental analyzer (PerkineElmer, Norwalk,
USA). The TLC was performed on Kieselgel 60 F254 (Merck) layer
using petroleum ether:ethyl acetate (3:1 v/v) as eluents.
For C15H14FN3S2 Calculated: C, 56.40; H, 4.42; N, 13.16. Found: C,
56.41; H, 4.40; N, 13.17.
MS (ESI) (m/z): (Mþþ1) 320.4.
4.1.1.3. 2-[5-(4-Fluorophenyl)-3-(5-chloro/methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-phenylthiazole
derivatives
(1e22).
A
mixture of 3-(5-chloro/methylthiophen-2-yl)-5-(4-
fluorophenyl)-1-thiocarbamoyl-2-pyrazoline (C/D) (0.001 mol)
and 2-bromoacetophenone/40-substituted-2-bromoacetophenone
(0.001 mol) in ethanol (20 mL) was refluxed for 6 h. The reaction
mixture was cooled and filtered.
4.1.1.3.1. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-phenylthiazole (1). Yield: 93%. Mp
149 ꢁC.
4.1.1. General procedure for the synthesis of the compounds
4.1.1.1. 1-(5-Chloro/methylthiophen-2-yl)-3-(4-fluorophenyl)prop-2-
en-1-ones (A/B). A mixture of 2-acetyl-5-chloro/methylthiophene
(0.05 mol), 4-fluorobenzaldehyde (0.05 mol) and 10% aqueous so-
dium hydroxide (10 mL) in ethanol (30 mL) was stirred at room
temperature for 10 h. The progress of the reaction was checked by
TLC. Upon completion, the reaction mixture was poured into
crushed ice. The precipitated solid was filtered, washed with water,
and dried. The product was crystallized from ethanol.
IR nmax (cmꢀ1): 3113.11, 3039.81 (aromatic CeH), 1600.92,
1535.34, 1508.33 (aromatic C]N and C]C stretching), 1444.68
(CeH bending), 1230.58, 1138.00, 1053.13, 1008.77 (CeN stretching
and aromatic CeH out of plane bending), 827.46, 792.74, 704.02
(aromatic CeH out of plane bending).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.38 (1H, dd,
JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.04 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.71 (1H, dd,
JMX ¼ 12 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 7.19e7.48 (10H, m,
aromatic protons), 7.70 (2H, dd, J ¼ 7.5 Hz, aromatic protons).
4.1.1.2. 3-(5-Chloro/methylthiophen-2-yl)-5-(4-fluorophenyl)-1-
thiocarbamoyl-2-pyrazolines (C/D). A mixture of chalcone (A/B)
(0.01 mol), thiosemicarbazide (0.012 mol) and sodium hydroxide
(0.01 mol) was refluxed in ethanol (25 mL) for 8 h. The solution was
poured into crushed ice. The precipitate was filtered and
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.52 (CH2), 64.38 (CH),
105.09 (CH), 115.67 and 115.89 (2CH), 125.93 (2CH), 128.01 (C),
128.40 (C), 128.94 (2CH), 129.22 (CH), 129.30 (CH), 129.82 (CH),
131.43 (CH), 133.50 (CH), 134.81 (C), 138.02 and 138.05 (C), 148.54

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
347
(C), 150.93 (C), 160.76 and 163.18 (C), 164.38 (C).
For C22H15ClFN3S2 Calculated: C, 60.06; H, 3.44; N, 9.55. Found:
C, 60.04; H, 3.43; N, 9.56.
127.79 (C), 128.67 and 128.75 (2CH), 129.24 (C), 130.97 (C), 131.29
(2CH), 132.88 (CH), 133.46 (CH), 137.31 (C), 148.09 (C), 149.18 (C),
160.21 and 162.63 (C), 163.94 (C).
MS (ESI) (m/z): (Mþþ1) 440.9.
For C22H14BrClFN3S2 Calculated: C, 50.93; H, 2.72; N, 8.10.
Found: C, 50.91; H, 2.74; N, 8.10.
4.1.1.3.2. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-fluorophenyl)thiazole (2). Yield: 94%.
Mp 159 ꢁC.
MS (ESI) (m/z): (Mþþ1) 519.8.
4.1.1.3.5. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-nitrophenyl)thiazole (5). Yield: 95%.
Mp 212 ꢁC.
IR nmax (cmꢀ1): 3113.11 (aromatic CeH), 2972.31 (aliphatic
CeH), 1604.77, 1539.20, 1521.84, 1508.33, 1485.19 (C]N and C]C
stretching), 1444.68 (CeH bending), 1313.52, 1220.94, 1155.36,
1047.35, 1010.70 (CeN stretching and aromatic CeH out of plane
bending), 827.46, 800.46, 732.95 (aromatic CeH out of plane
bending).
IR nmax (cmꢀ1): 3103.46, 3045.60 (aromatic CeH), 1595.13,
1546.91, 1506.41 (C]N and C]C stretching), 1446.61, 1328.95 (CeH
bending), 1224.80, 1132.21, 1053.13, 1008.77 (CeN stretching and
aromatic CeH out of plane bending), 852.54, 829.39, 794.67, 707.88
(aromatic CeH out of plane bending).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.38 (1H, dd,
JAM ¼ 18.0 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.02 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.70 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 7.19 (2H, dd,
J ¼ 8.5 Hz), 7.23e7.75 (9H, m, aromatic protons).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.40 (1H, dd,
JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.03 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.72 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 7.20e7.24 (3H, m,
aromatic protons), 7.35 (1H, d, J ¼ 3.5 Hz, aromatic proton),
7.47e7.50 (2H, m, aromatic protons), 7.72 (1H, s, thiazole), 7.95 (2H,
d, J ¼ 8.5 Hz, aromatic protons), 8.22 (2H, d, J ¼ 8.5 Hz, aromatic
protons).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.05 (CH2), 63.86 (CH),
104.34 (CH), 115.22 and 115.43 (4CH), 127.37 and 127.45 (2CH),
127.93 (C), 128.74 and 128.82 (2CH), 129.42 (CH), 130.92 and 130.95
(C),131.00 (2C),132.97 (CH),137.51 and 137.54 (C),148.15 (C),149.39
(C), 160.27e160.37 and 162.70e162.80 (C), 163.97 (C).
For C22H14ClF2N3S2 Calculated: C, 57.70; H, 3.08; N, 9.18. Found:
C, 57.72; H, 3.07; N, 9.18.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.00 (CH2), 63.81 (CH),
109.36 (CH), 115.21 and 115.43 (2CH), 123.92 (2CH), 126.19 (2CH),
127.93 (C),128.85 and 128.92 (2CH),129.59 (CH),131.11 (CH),132.79
(C), 137.17 and 137.21 (C), 140.23 (C), 146.18 (C), 148.32 (C), 148.59
(C), 160.28 and 162.71 (C), 164.14 (C).
MS (ESI) (m/z): (Mþþ1) 458.9.
4.1.1.3.3. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-chlorophenyl)thiazole
(3). Yield:
For C22H14ClFN4O2S2 Calculated: C, 54.49; H, 2.91; N, 11.55.
Found: C, 54.50; H, 2.90; N, 11.54.
88%. Mp 171 ꢁC.
IR nmax (cmꢀ1): 3138.18 (aromatic CeH), 2891.30 (aliphatic
CeH), 1575.84, 1537.27, 1475.54 (C]N and C]C stretching),
1446.61, 1369.46 (CeH bending), 1294.24, 1269.16, 1220.94,
1085.92, 1053.13 (CeN stretching and aromatic CeH out of plane
bending), 817.82, 788.89, 734.88 (aromatic CeH out of plane
bending).
MS (ESI) (m/z): (Mþþ1) 485.9.
4.1.1.3.6. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-cyanophenyl)thiazole (6). Yield: 72%.
Mp 204 ꢁC.
IR nmax (cmꢀ1): 3118.90 (aromatic CeH), 2987.74, 2900.94
(aliphatic CeH), 2223.92 (C^N stretching), 1604.77, 1544.98,
1508.33, 1452.40 (C]N and C]C stretching), 1408.04 (CeH
bending), 1311.59, 1215.15, 1047.35, 1014.56 (CeN stretching and
aromatic CeH out of plane bending), 833.25, 785.03, 740.67, 702.09
(aromatic CeH out of plane bending).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.38 (1H, dd,
JAM ¼ 18.0 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.00 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 11.5 Hz, C4-HM pyrazoline), 5.70 (1H, dd,
JMX ¼ 12 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 7.14e7.48 (9H, m,
aromatic protons), 7.71 (2H, d, J ¼ 8.5 Hz, aromatic protons).
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 3.39 (1H, dd, JAM ¼ 18.0 Hz,
13C NMR (100 MHz, DMSO-d6)
d
(ppm): 43.05 (CH2), 63.81 (CH),
JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.01 (1H, dd, JMA ¼ 17.5 Hz,
JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.71 (1H, dd, JMX ¼ 12.0 Hz,
JAX ¼ 7.0 Hz, C5-HX pyrazoline), 7.18e7.22 (3H, m, aromatic protons),
7.33 (1H, d, J ¼ 3.5 Hz, aromatic proton), 7.44e7.48 (2H, m, aromatic
protons), 7.63 (1H, s, thiazole), 7.81 (2H, d, J ¼ 8.5 Hz, aromatic pro-
tons), 7.87 (2H, d, J ¼ 8.5 Hz, aromatic protons).
105.41 (CH), 115.23 and 115.45 (2CH), 127.12 (2CH), 127.98 (C),
128.51 (2CH), 128.77 and 128.84 (2CH), 129.53 (CH), 131.00 (C),
131.97 (C), 132.93 (CH), 133.15 (C), 137.44 and 137.47 (C), 148.29 (C),
149.16 (C), 160.27 and 162.69 (C), 163.97 (C).
For C22H14Cl2FN3S2 Calculated: C, 55.70; H, 2.97; N, 8.86. Found:
C, 55.69; H, 2.95; N, 8.86.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.11 (CH2), 63.80 (CH),
MS (ESI) (m/z): (Mþþ1) 475.4.
108.44 (CH), 109.62 (C), 115.26 and 115.48 (2CH), 118.90 (C), 126.00
(2CH),128.00 (CH),128.76 and 128.84 (2CH),129.66 (CH),131.10 (C),
132.59 (2CH), 132.84 (C), 137.35 and 137.38 (C), 138.37 (C), 148.56
(C), 148.68 (C), 160.28 and 162.71 (C), 164.09 (C).
4.1.1.3.4. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-bromophenyl)thiazole
90%. Mp 191 ꢁC.
(4). Yield:
IR nmax (cmꢀ1): 3132.40, 3055.24 (aromatic CeH), 1604.77,
1535.34, 1508.33 (C]N and C]C stretching), 1448.54, 1398.39
(CeH bending), 1294.24,1213.23, 1155.36, 1099.43, 1051.20, 1006.84
(CeN stretching and aromatic CeH out of plane bending), 827.46,
788.89, 732.95 (aromatic CeH out of plane bending).
For C23H14ClFN4S2 Calculated: C, 59.41; H, 3.03; N, 12.05. Found:
C, 59.40; H, 3.03; N, 12.06.
MS (ESI) (m/z): (Mþþ1) 465.9.
4.1.1.3.7. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-methylsulfonylphenyl)thiazole
Yield: 67%. Mp 254 ꢁC.
(7).
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 3.38 (1H, dd,
JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.70 (1H, dd,
JMX ¼ 12 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 7.17e7.35 (4H, m, ar-
omatic protons), 7.41 (1H, s, thiazole), 7.44e7.47 (2H, m, aromatic
protons), 7.55 (2H, d, J ¼ 8.5 Hz, aromatic protons), 7.65 (2H, d,
J ¼ 8.5 Hz, aromatic protons).
IR nmax (cmꢀ1): 3113.11, 3039.81 (aromatic CeH), 2931.80
(aliphatic CeH), 1593.20, 1531.48, 1508.33 (C]N and C]C
stretching), 1404.18 (CeH bending), 1300.02, 1278.81, 1213.23,
1147.65, 1051.20, 1008.77 (CeN stretching and aromatic CeH out of
plane bending), 964.41, 833.25, 773.46, 732.95 (aromatic CeH out
of plane bending).
13C NMR (100 MHz, DMSO-d6)
d
(ppm): 42.93 (CH2), 63.80 (CH),
1H NMR (500 MHz, DMSO-d6)
(1H, dd, JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.03 (1H, dd,
d (ppm): 3.21 (3H, s, SO2CH3), 3.38
105.37 (CH), 115.10 and 115.31 (2CH), 120.48 (C), 127.34 (2CH),

348
M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.72 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 7.20e7.23 (3H, m,
aromatic protons), 7.34 (1H, d, J ¼ 4 Hz, aromatic proton), 7.47e7.50
(2H, m, aromatic protons), 7.63 (1H, s, thiazole), 7.90 (2H, d,
J ¼ 8.5 Hz, aromatic protons), 7.94 (2H, d, J ¼ 8.5 Hz, aromatic
protons).
1604.77, 1543.05, 1508.33, 1477.47 (C]N and C]C stretching),
1446.61, 1354.03 (CeH bending), 1307.74, 1222.87, 1107.14, 1035.77
(CeN stretching and aromatic CeH out of plane bending), 939.33,
835.18, 790.81, 732.95 (aromatic CeH out of plane bending).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.14 (1H, dd,
JAM ¼ 17.5 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.00 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.68 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 6.02 (2H, s,
OeCH2eO), 6.88 (1H, m, aromatic proton), 7.15e7.23 (6H, m, aro-
matic protons), 7.31 (1H, d, J ¼ 4.0 Hz, aromatic proton), 7.44e7.47
(2H, m, aromatic protons).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 42.95 (CH2), 43.49 (CH3),
63.87 (CH), 108.03 (CH), 115.14 and 115.35 (2CH), 125.93 (2CH),
127.27 (2CH), 127.88 (CH), 128.81 and 128.90 (2CH), 129.45 (CH),
131.03 (C), 132.83 (C), 137.21 and 137.24 (C), 138.75 (C), 139.23 (C),
148.43 (C), 148.68 (C), 160.24 and 162.67 (C), 164.08 (C).
For C23H17ClFN3O2S3 Calculated: C, 53.32; H, 3.31; N, 8.11.
Found: C, 53.30; H, 3.30; N, 8.14.
13C NMR (100 MHz, DMSO-d6)
d (ppm): 43.02 (CH2), 63.91 (CH),
100.97 (CH2), 103.06 (CH), 105.79 (CH), 108.21 (CH), 115.16 and
115.37 (2CH),119.30 (CH),127.88 (C),128.68 (CH),128.76 and 128.81
(2CH), 129.27 (CH), 130.92 (C), 133.00 (C), 137.57 and 137.60 (C),
146.68 (C), 147.41 (C), 147.97 (C), 150.09 (C), 160.24 and 162.66 (C),
163.63 (C).
MS (ESI) (m/z): (Mþþ1) 519.0.
4.1.1.3.8. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-methylphenyl)thiazole
68%. Mp 166 ꢁC.
(8). Yield:
IR nmax (cmꢀ1): 3140.11, 3043.67 (aromatic CeH), 2910.58
(aliphatic CeH), 1633.71, 1577.77, 1523.76, 1508.33, 1487.12 (C]N
and C]C stretching), 1446.61, 1369.46 (CeH bending), 1294.24,
1219.01, 1211.30, 1051.20 (CeN stretching and aromatic CeH out of
plane bending), 829.39, 817.82, 786.96, 731.02 (aromatic CeH out of
plane bending).
For C23H15ClFN3O2S2 Calculated: C, 57.08; H, 3.12; N, 8.68.
Found: C, 57.07; H, 3.11; N, 8.68.
MS (ESI) (m/z): (Mþþ1) 484.9.
4.1.1.3.11. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-trifluoromethylphenyl)thiazole (11).
Yield: 77%. Mp 176 ꢁC.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.28 (3H, s, CH3), 3.32
IR nmax (cmꢀ1): 3115.04 (aromatic CeH), 1606.70, 1546.91,
1525.69, 1508.33, 1450.47 (C]N and C]C stretching), 1411.89 (CeH
bending), 1323.17, 1309.67, 1224.80, 1153.43, 1103.28, 1066.64,
1008.77 (CeN stretching and aromatic CeH out of plane bending),
827.46 (aromatic CeH out of plane bending), 698.23 (CeS
stretching).
(1H, dd, JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 3.98 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.68 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 7.14e7.47 (9H, m,
aromatic protons), 7.59 (2H, d, J ¼ 8.0 Hz, aromatic protons).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 20.64 (CH3), 42.91 (CH2),
63.85 (CH), 103.58 (CH), 115.07 and 115.28 (2CH), 125.32 (2CH),
127.81 (CH), 128.67 and 128.75 (2CH), 128.93 (2CH), 129.16 (CH),
130.83 (C), 131.64 (C), 132.99 (C), 136.73 (C), 137.46 and 137.49 (C),
147.87 (C), 150.47 (C), 160.20 and 162.62 (C), 163.74 (C).
For C23H17ClFN3S2 Calculated: C, 60.85; H, 3.77; N, 9.26. Found:
C, 60.85; H, 3.76; N, 9.27.
1H NMR (500 MHz, DMSO-d6)
d (ppm): 3.38 (1H, dd,
JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.71 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 7.18e7.24 (3H, m,
aromatic protons), 7.32 (1H, d, J ¼ 4.0 Hz, aromatic proton),
7.46e7.49 (2H, m, aromatic protons), 7.56 (1H, s, thiazole), 7.70 (2H,
d, J ¼ 8.0 Hz, aromatic protons), 7.90 (2H, d, J ¼ 8.0 Hz, aromatic
protons).
MS (ESI) (m/z): (Mþþ1) 454.9.
4.1.1.3.9. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-methoxyphenyl)thiazole
Yield: 69%. Mp 209 ꢁC.
(9).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 42.93 (CH2), 63.86 (CH),
107.21 (CH), 115.10 and 115.31 (2CH), 122.82 (C), 125.28 and 125.32
(CH), 125.52 (C), 125.86 (2CH), 127.73 (CH), 128.72 and 128.80
(2CH), 129.26 (CH), 131.07 (C), 132.87 (CH), 137.21 and 137.23 (C),
137.92 (C),148.20 and 148.23 (C),148.86 (2C),160.26 and 162.68 (C),
164.08 (C).
IR nmax (cmꢀ1): 3109.25 (aromatic CeH), 2937.59 (aliphatic
CeH), 1608.63, 1539.20, 1519.91, 1506.41, 1485.19 (C]N and C]C
stretching), 1442.75 (CeH bending), 1286.52, 1249.87, 1219.01,
1170.79, 1051.20, 1029.99, 1008.77 (CeN stretching and aromatic
CeH out of plane bending), 829.39, 798.53, 736.81 (aromatic CeH
out of plane bending).
For C23H14ClF4N3S2 Calculated: C, 54.38; H, 2.78; N, 8.27. Found:
C, 54.36; H, 2.80; N, 8.25.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 3.32 (1H, dd,
MS (ESI) (m/z): (Mþþ1) 508.9.
JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 3.76 (3H, s, OCH3),
3.99 (1H, dd, JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.68
(1H, dd, JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 6.90 (2H, d,
J ¼ 8.5 Hz, aromatic protons), 7.15 (1H, s, thiazole), 7.18e7.21 (3H, m,
aromatic protons), 7.30 (1H, d, J ¼ 4.0 Hz, aromatic proton),
7.44e7.47 (2H, m, aromatic protons), 7.63 (2H, d, J ¼ 9.0 Hz, aro-
matic protons).
4.1.1.3.12. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-phenylthiazole (12). Yield: 87%. Mp
146 ꢁC.
IR nmax (cmꢀ1): 3113.11, 3064.89 (aromatic CeH), 2914.44
(aliphatic CeH), 1600.92, 1537.27, 1508.33, 1475.54 (C]N and C]C
stretching), 1442.75, 1381.03 (CeH bending), 1232.51, 1138.00,
1051.20, 1026.13 (CeN stretching and aromatic CeH out of plane
bending), 842.89, 825.53, 796.60, 771.53, 705.95 (aromatic CeH out
of plane bending).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 42.93 (CH2), 55.00 (CH3),
63.87 (CH), 102.36 (CH), 113.79 (2CH), 115.09 and 115.30 (2CH),
126.72 (2CH), 127.18 (CH), 127.83 (CH), 128.66 and 128.74 (2CH),
129.17 (C), 130.81 (C), 133.00 (C), 137.51 and 137.54 (C), 147.84 (C),
150.26 (C), 158.74 (C), 160.19 and 162.62 (C), 163.75 (C).
For C23H17ClFN3OS2 Calculated: C, 58.78; H, 3.65; N, 8.94. Found:
C, 58.76; H, 3.66; N, 8.95.
1H NMR (500 MHz, DMSO-d6)
d (ppm): 2.51 (3H, s, CH3), 3.32
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 11.5 Hz, C4-HM pyrazoline), 5.67 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.87 (1H, m, aromatic
proton), 7.18e7.36 (7H, m, aromatic protons), 7.44e7.47 (2H, m,
aromatic protons), 7.70 (2H, d, J ¼ 8.5 Hz, aromatic protons).
MS (ESI) (m/z): (Mþþ1) 470.9.
4.1.1.3.10. 2-[5-(4-Fluorophenyl)-3-(5-chlorothiophen-2-yl)-4,5-
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.10 (CH3), 43.43 (CH2),
dihydro-1H-pyrazol-1-yl]-4-(1,3-benzodioxol-5-yl)thiazole
(10).
63.56 (CH), 104.18 (CH), 115.07 and 115.29 (2CH), 125.37 (2CH),
126.33 (C), 127.38 (CH), 128.35 (2CH), 128.57 (CH), 128.65 (CH),
129.80 (CH), 131.57 (CH), 134.37 (C), 137.68 and 137.71 (C), 143.08
Yield: 73%. Mp 185 ꢁC.
IR nmax (cmꢀ1): 3111.18 (aromatic CeH), 2895.15 (aliphatic CeH),

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
349
(C), 148.77 (C), 150.37 (C), 160.16 and 162.58 (C), 164.12 (C).
For C23H18FN3S2 Calculated: C, 65.85; H, 4.32; N, 10.02. Found: C,
65.85; H, 4.30; N, 10.03.
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.86 (1H, m, aromatic
proton), 7.19 (2H, m, aromatic protons), 7.24 (1H, d, J ¼ 3.0 Hz, ar-
omatic proton), 7.37 (1H, s, thiazole), 7.43e7.45 (2H, m, aromatic
protons), 7.54 (2H, d, J ¼ 8.5 Hz, aromatic protons), 7.65 (2H, d,
J ¼ 8.5 Hz, aromatic protons).
MS (ESI) (m/z): (Mþþ1) 420.5.
4.1.1.3.13. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-fluorophenyl)thiazole
Yield: 88%. Mp 168 ꢁC.
(13).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.09 (CH3), 43.41 (CH2),
63.51 (CH), 105.04 (CH), 115.08 and 115.29 (2CH), 120.41 (C), 126.31
(C), 127.34 (2CH), 128.58 and 128.66 (2CH), 129.85 (C), 131.29 (2CH),
131.50 (CH), 133.55 (CH), 137.52 and 137.55 (C), 143.14 (C), 148.91
(C), 149.16 (C), 160.16 and 162.58 (C), 164.21 (C).
IR nmax (cmꢀ1): 3111.18, 3055.24 (aromatic CeH), 2927.24
(aliphatic CeH), 1591.27, 1535.34, 1508.33, 1481.33 (C]N and C]C
stretching), 1313.52, 1276.88, 1217.08, 1153.43, 1112.93, 1043.49
(CeN stretching and aromatic CeH out of plane bending), 827.46,
802.39, 732.95 (aromatic CeH out of plane bending), 696.30 (CeS
stretching).
For C23H17BrFN3S2 Calculated: C, 55.42; H, 3.44; N, 8.43. Found:
C, 55.41; H, 3.45; N, 8.43.
MS (ESI) (m/z): (Mþþ1) 499.4.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.52 (3H, s, CH3), 3.32
4.1.1.3.16. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-nitrophenyl)thiazole (16). Yield: 83%.
Mp 210 ꢁC.
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.67 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.87 (1H, m, aromatic
proton), 7.17 (2H, d, J ¼ 9.0 Hz, aromatic protons), 7.21 (2H, d,
J ¼ 9.0 Hz, aromatic protons), 7.24 (1H, d, J ¼ 3.5 Hz, aromatic
proton), 7.29 (1H, s, thiazole), 7.43e7.46 (2H, m, aromatic protons),
7.72e7.75 (2H, m, aromatic protons).
IR nmax (cmꢀ1): 3115.04 (aromatic CeH), 2918.30 (aliphatic
CeH), 1595.13, 1533.41, 1504.48 (C]N and C]C stretching),
1336.67, 1315.45, 1222.87, 1155.36, 1105.21, 1047.35 (CeN stretching
and aromatic CeH out of plane bending), 835.18, 790.81, 731.02
(aromatic CeH out of plane bending).
13C NMR (100 MHz, DMSO-d6)
d
(ppm): 15.09 (CH3), 43.44 (CH2),
1H NMR (500 MHz, DMSO-d6)
d (ppm): 2.51 (3H, s, CH3), 3.44
63.55 (CH), 103.90 (CH), 115.09 and 115.30 (4CH), 126.33 (C), 127.29
and 127.37 (2CH), 128.55 and 128.63 (2CH), 129.83 (CH), 130.97 and
131.00 (2C), 131.54 (CH), 137.63 and 137.66 (C), 143.12 (C), 148.84
(C), 149.32 (C), 160.16e160.28 and 162.58e162.71 (C), 164.22 (C).
For C23H17F2N3S2 Calculated: C, 63.14; H, 3.92; N, 9.60. Found: C,
63.13; H, 3.92; N, 9.61.
(1H, dd, JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.67 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.86 (1H, m, aromatic
proton), 7.21 (2H, m, aromatic protons), 7.25 (1H, d, J ¼ 3.5 Hz, ar-
omatic proton), 7.46 (2H, dd, J ¼ 8.5, 5.5 Hz, aromatic protons), 7.67
(1H, s, thiazole), 7.94 (2H, d, J ¼ 9.0 Hz, aromatic protons), 8.21 (2H,
d, J ¼ 9.0 Hz, aromatic protons).
MS (ESI) (m/z): (Mþþ1) 438.5.
4.1.1.3.14. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.09 (CH3), 43.43 (CH2),
dihydro-1H-pyrazol-1-yl]-4-(4-chlorophenyl)thiazole
(14).
63.52 (CH), 108.95 (CH), 115.13 and 115.34 (2CH), 123.82 (2CH),
126.12 (2CH), 126.34 (C), 128.69 and 128.77 (2CH), 130.02 (CH),
131.40 (CH), 137.35 and 137.38 (C), 140.29 (C), 143.31 (C), 146.11 (C),
148.30 (C), 149.26 (C), 160.22 and 162.64 (C), 164.37 (C).
For C23H17FN4O2S2 Calculated: C, 59.47; H, 3.69; N, 12.06. Found:
C, 59.46; H, 3.70; N, 12.05.
Yield: 75%. Mp 172 ꢁC.
IR nmax (cmꢀ1): 3095.75 (aromatic CeH), 2922.16 (aliphatic
CeH), 1604.77,1546.91,1508.33,1471.69 (C]N and C]C stretching)
1311.59, 1224.80, 1089.78, 1049.28, 1012.63 (CeN stretching and
aromatic CeH out of plane bending), 825.53, 802.39, 729.09 (aro-
matic CeH out of plane bending), 692.44 (CeS stretching).
MS (ESI) (m/z): (Mþþ1) 465.5.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.51 (3H, s, CH3), 3.32
4.1.1.3.17. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.67 (1H, dd,
JMX ¼ 12.0 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.86 (1H, m, aromatic
proton), 7.20 (2H, m, aromatic protons), 7.25 (1H, d, J ¼ 3.5 Hz,
aromatic proton), 7.38 (1H, s, thiazole), 7.41 (2H, d, J ¼ 8.5 Hz, ar-
omatic protons), 7.44e7.46 (2H, m, aromatic protons), 7.72 (2H, d,
J ¼ 8.5 Hz, aromatic protons).
dihydro-1H-pyrazol-1-yl]-4-(4-cyanophenyl)thiazole
(17).
Yield: 84%. Mp 203 ꢁC.
IR nmax (cmꢀ1): 3115.04 (aromatic CeH), 2970.38, 2918.30
(aliphatic CeH), 2218.14 (C^N stretching), 1606.70, 1539.20,
1508.33, 1479.40 (C]N and C]C stretching), 1301.95, 1222.87,
1155.36, 1047.35 (CeN stretching and aromatic CeH out of plane
bending), 835.18, 792.74,740.67 (aromatic CeH out of plane
bending).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.09 (CH3), 43.43 (CH2),
63.51 (CH), 104.96 (CH), 115.09 and 115.30 (2CH), 126.33 (C), 127.04
(2CH), 128.38 (2CH), 128.57 and 128.65 (2CH), 129.88 (C), 131.50
(CH), 131.84 (CH), 133.21 (C), 137.55 and 137.58 (C), 143.16 (C),
148.94 (C), 149.11 (C), 160.16 and 162.58 (C), 164.22 (C).
For C23H17ClFN3S2 Calculated: C, 60.85; H, 3.77; N, 9.26. Found:
C, 60.86; H, 3.75; N, 9.25.
1H NMR (500 MHz, DMSO-d6)
d (ppm): 2.54 (3H, s, CH3), 3.34
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.01 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.67 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.86 (1H, m, aromatic
proton), 7.19 (2H, m, aromatic protons), 7.24 (1H, d, J ¼ 3.5 Hz, ar-
omatic proton), 7.45 (2H, dd, J ¼ 8.5, 5.5 Hz, aromatic protons), 7.60
(1H, s, thiazole), 7.80 (2H, d, J ¼ 8.5 Hz, aromatic protons), 7.87 (2H,
d, J ¼ 8.5 Hz, aromatic protons).
MS (ESI) (m/z): (Mþþ1) 454.9.
4.1.1.3.15. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-bromophenyl)thiazole
Yield: 85%. Mp 194 ꢁC.
(15).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.09 (CH3), 43.47 (CH2),
63.49 (CH), 107.97 (CH), 109.50 (C), 115.12 and 115.33 (2CH), 118.80
(C), 125.92 (2CH), 126.33 (C), 128.56 and 128.64 (2CH), 129.97 (CH),
131.42 (CH), 132.42 (2CH), 137.44 and 137.48 (C), 138.40 (C), 143.27
(C), 148.62 (C), 149.16 (C), 160.18 and 162.60 (C), 164.32 (C).
For C24H17FN4S2 Calculated: C, 64.84; H, 3.85; N, 12.60. Found: C,
64.85; H, 3.83; N, 12.60.
IR nmax (cmꢀ1): 3132.40, 3043.67 (aromatic CeH), 2887.44
(aliphatic CeH), 1604.77, 1531.48, 1508.33, 1471.69 (C]N and C]C
stretching), 1398.39 (CeH bending), 1219.01, 1155.36, 1097.50,
1045.42, 1006.84 (CeN stretching and aromatic CeH out of plane
bending), 819.75, 804.32, 790.81, 732.95 (aromatic CeH out of
plane bending).
MS (ESI) (m/z): (Mþþ1) 445.5.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.51 (3H, s, CH3), 3.32
4.1.1.3.18. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-methylsulfonylphenyl)thiazole (18).
Yield: 70%. Mp 241 ꢁC.
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.00 (1H, dd,
JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.66 (1H, dd,

350
M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
IR nmax (cmꢀ1): 3113.11, 3016.67 (aromatic CeH), 2953.02
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.12 (CH3), 43.42 (CH2),
(aliphatic CeH), 1593.20, 1529.55, 1508.33 (C]N and C]C
stretching), 1404.18 (CeH bending), 1298.09, 1278.81, 1213.23,
1147.65, 1087.85, 1049.28 (CeN stretching and aromatic CeH out of
plane bending), 958.62, 835.18, 798.53, 773.46, 731.02 (aromatic
CeH out of plane bending).
55.01 (CH3), 63.61 (CH), 102.05 (CH), 113.79 (2CH), 115.09 and
115.30 (2CH), 126.33 (CH), 126.74 (2CH), 127.31 (CH), 128.60 and
128.68 (2CH), 129.75 (C), 131.64 (C), 137.75 and 137.78 (C), 143.05
(C), 148.64 (C), 150.26 (C), 158.72 (C), 160.17 and 162.59 (C), 164.07
(C).
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.51 (3H, s, CH3), 3.21
For C24H20FN3OS2 Calculated: C, 64.12; H, 4.48; N, 9.35. Found:
C, 64.14; H, 4.45; N, 9.34.
(3H, s, SO2CH3), 3.37 (1H, dd, JAM ¼ 18.0 Hz, JAX ¼ 7.0 Hz, C4-HA
pyrazoline), 4.02 (1H, dd, JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM
pyrazoline), 5.68 (1H, dd, JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyr-
azoline), 6.87 (1H, m, aromatic proton), 7.21 (2H, m, aromatic
protons), 7.26 (1H, d, J ¼ 3.5 Hz, aromatic proton), 7.47 (2H, dd,
J ¼ 8.5, 5.5 Hz, aromatic protons), 7.60 (1H, s, thiazole), 7.90 (2H, d,
J ¼ 8.5 Hz, aromatic protons), 7.95 (2H, d, J ¼ 8.5 Hz, aromatic
protons).
MS (ESI) (m/z): (Mþþ1) 450.5.
4.1.1.3.21. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(1,3-benzodioxol-5-yl)thiazole
Yield: 82%. Mp 205 ꢁC.
(21).
IR nmax (cmꢀ1): 3116.97 (aromatic CeH), 2972.31, 2900.94
(aliphatic CeH), 1602.85, 1544.98, 1508.33, 1479.40 (C]N and C]C
stretching), 1357.89 (CeH bending), 1303.88, 1251.80, 1222.87,
1103.28, 1039.63 (CeN stretching and aromatic CeH out of plane
bending), 835.18, 792.74, 738.74 (aromatic CeH out of plane
bending).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.25 (CH3), 43.50 (CH2),
43.54 (CH3), 63.60 (CH), 107.83 (CH), 115.24 and 115.45 (2CH),
126.00 (2CH), 126.49 (C), 127.39 (2CH), 128.83 and 128.92 (2CH),
130.23 (CH), 131.49 (CH), 137.53 and 137.57 (C), 138.90 (C), 139.19
(C), 143.39 (C), 148.72 (C), 149.34 (C), 160.27 and 162.69 (C), 164.35
(C).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 2.54 (3H, s, CH3), 3.29
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 8.0 Hz, C4-HA pyrazoline), 3.98 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline), 5.64 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.02 (2H, s,
OeCH2eO), 6.82e6.89 (2H, m, aromatic protons), 7.08e7.27 (6H, m,
aromatic protons), 7.44 (2H, dd, J ¼ 8.0, 5.5 Hz, aromatic protons).
For C24H20FN3O2S3 Calculated: C, 57.93; H, 4.05; N, 8.44. Found:
C, 57.92; H, 4.04; N, 8.46.
MS (ESI) (m/z): (Mþþ1) 498.6.
4.1.1.3.19. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.12 (CH3), 43.46 (CH2),
dihydro-1H-pyrazol-1-yl]-4-(4-methylphenyl)thiazole
(19).
63.62 (CH), 100.91 (CH2), 102.71 (CH), 105.79 (CH), 108.16 (CH),
115.09 and 115.31 (2CH), 119.26 (CH), 126.34 (C), 128.56 and 128.64
(2CH), 128.91 (C), 129.79 (CH), 131.60 (CH), 137.76 and 137.79 (C),
143.09 (C), 146.63 (C), 147.37 (C), 148.73 (C), 150.06 (C), 160.17 and
162.60 (C), 163.92 (C).
Yield: 73%. Mp 178 ꢁC.
IR nmax (cmꢀ1): 3140.11, 3057.17 (aromatic CeH), 2912.51
(aliphatic CeH), 1604.77, 1523.76, 1508.33, 1481.33 (C]N and C]C
stretching), 1294.24, 1219.01, 1155.36, 1097.50, 1045.42 (CeN
stretching and aromatic CeH out of plane bending), 831.32, 817.82,
790.81, 732.95 (aromatic CeH out of plane bending).
For C24H18FN3O2S2 Calculated: C, 62.19; H, 3.91; N, 9.06. Found:
C, 62.20; H, 3.90; N, 9.05.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.29 (3H, s, CH3), 2.51
MS (ESI) (m/z): (Mþþ1) 464.5.
(3H, s, CH3), 3.31 (1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyr-
azoline), 3.98 (1H, dd, JMA ¼ 17.5 Hz, JMX ¼ 12.0 Hz, C4-HM pyr-
azoline), 5.65 (1H, dd, JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline),
6.85 (1H, m, aromatic proton), 7.14e7.23 (6H, m, aromatic protons),
7.44 (2H, dd, J ¼ 8.5, 5.5 Hz, aromatic protons), 7.60 (2H, d,
J ¼ 8.0 Hz, aromatic protons).
4.1.1.3.22. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-trifluoromethylphenyl)thiazole (22).
Yield: 68%. Mp 158 ꢁC.
IR nmax (cmꢀ1): 3111.18 (aromatic CeH), 2920.23 (aliphatic
CeH), 1612.49, 1550.77, 1525.69, 1512.19 (C]N and C]C stretch-
ing), 1323.17, 1234.44, 1157.29, 1114.86, 1066.64, 1014.56 (CeN
stretching and aromatic CeH out of plane bending), 829.39, 848.68,
804.32, 705.95 (aromatic CeH out of plane bending).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.09 (CH3), 20.64 (CH3),
43.39 (CH2), 63.57 (CH), 103.24 (CH), 115.04 and 115.25 (2CH),
125.32 (2CH), 126.31 (C), 128.58 and 128.66 (2CH), 128.92 (2CH),
129.31 (CH), 131.60 (CH), 131.74 (C), 136.66 (C), 137.69 and 137.71
(C), 143.03 (C), 148.66 (C), 150.45 (C), 160.15 and 162.57 (C), 164.05
(C).
1H NMR (500 MHz, DMSO-d6)
d (ppm): 2.51 (3H, s, CH3), 3.37
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 6.5 Hz, C4-HA pyrazoline), 4.02 (1H, dd,
JMA ¼ 17.5 Hz, JMX ¼ 11.5 Hz, C4-HM pyrazoline), 5.69 (1H, dd,
JMX ¼ 11.5 Hz, JAX ¼ 6.5 Hz, C5-HX pyrazoline), 6.87 (1H, m, aromatic
proton), 7.21 (2H, m, aromatic protons), 7.26 (1H, d, J ¼ 3.5 Hz, ar-
omatic proton), 7.46 (2H, dd, J ¼ 8.5, 5.5 Hz, aromatic protons), 7.56
(1H, s, thiazole), 7.72 (2H, d, J ¼ 8.0 Hz, aromatic protons), 7.91 (2H,
d, J ¼ 8.5 Hz, aromatic protons).
For C24H20FN3S2 Calculated: C, 66.49; H, 4.65; N, 9.69. Found: C,
66.50; H, 4.64; N, 9.68.
MS (ESI) (m/z): (Mþþ1) 434.5.
4.1.1.3.20. 2-[5-(4-Fluorophenyl)-3-(5-methylthiophen-2-yl)-4,5-
dihydro-1H-pyrazol-1-yl]-4-(4-methoxyphenyl)thiazole
Yield: 78%. Mp 186 ꢁC.
(20).
13C NMR (100 MHz, DMSO-d6)
d (ppm): 15.23 (CH3), 43.52 (CH2),
63.57 (CH), 107.08 (CH), 115.23 and 115.45 (2CH), 122.95 (C), 125.47
and 125.51 (CH), 125.65 (C), 125.96 (2CH), 126.48 (CH), 127.39 (C),
128.76 and 128.85 (2CH),130.18 (CH),131.52 (CH),137.58 and 137.61
(C), 138.09 (C), 143.36 (C), 148.87 (C), 149.25 (C), 160.27 and 162.69
(C), 164.37 (C).
IR nmax (cmꢀ1): 3109.25 (aromatic CeH), 2929.87, 2839.22
(aliphatic CeH), 1606.70, 1521.84, 1508.33, 1485.19 (C]N and C]C
stretching), 1317.38, 1286.52, 1246.02, 1215.15, 1170.79, 1047.35,
1028.06 (CeN stretching and aromatic CeH out of plane bending),
829.39, 804.32, 736.81, 698.23 (aromatic CeH out of plane
bending).
For C24H17F4N3S2 Calculated: C, 59.13; H, 3.51; N, 8.62. Found: C,
59.11; H, 3.53; N, 8.62.
1H NMR (500 MHz, DMSO-d6)
d
(ppm): 2.51 (3H, s, CH3), 3.28
MS (ESI) (m/z): (Mþþ1) 488.5.
(1H, dd, JAM ¼ 17.5 Hz, JAX ¼ 7.5 Hz, C4-HA pyrazoline), 3.76 (3H, s,
OCH3), 3.99 (1H, dd, JMA ¼ 18.0 Hz, JMX ¼ 12.0 Hz, C4-HM pyrazoline),
5.65 (1H, dd, JMX ¼ 12.0 Hz, JAX ¼ 7.0 Hz, C5-HX pyrazoline), 6.85 (1H,
m, aromatic protons), 6.91 (2H, d, J ¼ 9.0 Hz, aromatic proton), 7.13
(1H, s, thiazole), 7.15e7.24 (3H, m, aromatic protons), 7.44 (2H, dd,
J ¼ 8.5, 6.0 Hz, aromatic protons), 7.63 (2H, d, J ¼ 9.0 Hz, aromatic
protons).
4.2. Microbiology
The antifungal assay was carried out according to the micro-
broth dilution method with some modifications [36,37]. The com-
pounds (1e22) were tested for their in vitro antifungal activity
against C. glabrata (Clinical Isolate, Osmangazi University, Faculty of

M.D. Altıntop et al. / European Journal of Medicinal Chemistry 92 (2015) 342e352
351
Medicine, Eskis¸ ehir, Turkey), Candida albicans (ATCC 90028), C.
tropicalis (NRLL Y-12968), C. krusei (NRLL Y-7179), C. zeylanoides
(NRLLY-1774), Candida parapsilosis (NRLLY-12696), A. flavus (NRRL-
980), A. niger (ATCC-1094), Aspergillus parasiticus (NRRL 465), F.
solani (NRRL-13414).
Microbroth dilution-susceptibility assay was used for antifungal
evaluation of the compounds. The stock solutions of the samples
were prepared in dimethyl sulfoxide (DMSO, Merck). Dilution se-
ries using sterile distilled water were prepared from 4 mg/mL to
0.0039 mg/mL in micro-test tubes that were transferred to 96-well
microtiter plates. Fungal strains grown on Potato Dextrose Agar
(PDA) at 25 ꢁC for 5 days for molds and Sabouraud Dextrose Agar
(SDA) at 37 ꢁC for 24 h for yeasts suspensions in double-strength
Potato Dextrose Broth (PDB) were standardized to 105 spores/mL.
incubated in the environmental shaker at 37 ꢁC, 250 rpm for
90 min. Each sample was analyzed in triplicate. At the end of
90 min, 2.8 mL of indicator medium (Xenometrix AG, Gewerber-
trasse, Switzerland) was added to each well of the 24-well plates.
50 mL aliquots were distributed into 48 wells of a 384-well-plate
for each concentration and was incubated at 37 ꢁC in a dry incu-
bator for 48 h. The presence of the revertant bacteria dropped the
pH of solution resulting in color change of the indicator from
purple to yellow. The number of positive (yellow) wells out of 48
wells in triplicates were counted and compared with the negative
control. Fold induction over the negative control and fold induc-
tion over the baseline were calculated. Fold induction over the
negative control is the ratio of the mean number of positive wells
for the dose concentration divided by the mean number of posi-
tive wells for the zero dose (negative) control. Fold induction over
the baseline is the ratio of the mean number of positive wells for
the dose concentration divided by zero dose baseline. The zero
dose baseline is obtained by adding one standard deviation to the
mean number of positive wells of the zero dose control.
Mutagenity was determined according to the criteria from
previous studies [35] as follows:
100 mL of each spore suspension was then added into the wells. The
last well-chain without a fungus was used as a negative control.
Sterile distilled water and the medium served as a positive growth
control. After incubation at 25 ꢁC (for molds) for 72 h and 37 ꢁC (for
yeasts) for 48 h, antifungal activity was detected by the investiga-
tion of mycelia growing under stereo microscope for molds. After
incubation at 37 ꢁC for 48 h, antifungal activities against to yeasts
were detected by spraying of 0.5% triphenyl tetrazolium chloride
(TTC, Merck) aqueous solution. Minimum inhibitory concentration
(MIC) was defined as the lowest concentration of compounds that
inhibited visible growth, as indicated by the TTC staining. Ketoco-
nazole was used as an antifungal agent.
If the baseline was ꢂ3, significant increases between 2 and 3-
fold the baseline were classified as weak positive, and increases
of greater than threefold the baseline were classified as positive. If
the baseline was >3, significant increases between 1.5 and 2.5-fold
the baseline were classified as weak positive, and increases of
greater than 2.5-fold the baseline were classified as positive. To be
classified as a mutagenic compound, there should be at least two
adjacent doses with significant increases or a significant increase at
the highest dose level. All doses were tested for according to Stu-
dent's t-test at p < 0.05 for statistical significance. Compounds
which did not have any of above properties were classified as
negative.
4.3. Genotoxicity test
4.3.1. Ames MPF
Ames assay was carried out to determine the mutagenicity of
the compounds using Ames MPF 98/100 mutagenicity assay
sample kit (Xenometrix AG, Gewerbertrasse, Switzerland) [38].
Salmonella typhimurium strains, TA98 (frameshift mutations) and
TA100 (base-pair substitutions), were thawed and growth medium
(200 mL) was added to obtain homogenous suspensions of Sal-
monella strains (TA98 and TA100). The suspension (25 mL) was
4.4. Cytotoxicity
NIH/3T3 mouse embryonic fibroblast and A549 human lung
adenocarcinoma cell lines were used for cytotoxicity assay. NIH/3T3
cells were incubated in Dulbecco's modified Eagle's medium
(DMEM; Hyclone, Thermo Scientific, USA) supplemented with fetal
calf serum (Hyclone, Thermo Scientific, USA), 100 IU/mL penicillin
and 100 mg/mL streptomycin (Hyclone, Thermo Scientific, USA)
and 7.5% NaHCO3 at 37 ꢁC in a humidified atmosphere of 95% air
and 5% CO2. A549 cells were incubated in RPMI medium (Hyclone,
Thermo Scientific, USA) supplemented with fetal calf serum,100 IU/
mL penicillin and 100 mg/mL streptomycin and 7.5% NaHCO3 at
37 ꢁC in a humidified atmosphere of 95% air and 5% CO2. NIH/3T3
and A549 cells were seeded at 20,000 cells into each well of 96-well
plates. After 24 h of incubating period, the culture mediums were
removed and the compound was added to culture medium at
added to a mixture of 10 mL growth medium and 10 mL ampicillin
(50 mg/mL). Negative control, which was devoid of Salmonella
strains, was also prepared. The culture tubes were incubated in a
shaker (SI-600, Jeio Tech, Korea) at 37 ꢁC, 250 rpm for 14e16 h.
The ‘overnight grown’ cultures were diluted 1:10 with growth
medium and the absorbance was measured at 600 nm. The
absorbance for the ‘overnight grown’ culture and negative control
should be 0.25 and 0.005, respectively. Dose range for the chem-
icals with the tester strains according to the previous guidelines at
a concentration range of 16e5000 mg/mL [39]. Compounds 1, 3, 4,
11, 17 and 18 were prepared in six different concentrations (5, 2.5,
1.25, 0.625, 0.3125, 0.156 mg/mL) in DMSO. Mutagenic potential of
the compounds was determined in the absence and the presence
of Aroclor™-1254 induced male SpragueeDawley rat liver
microsomal enzyme (S9) mix (Xenometrix AG, Gewerbertrasse,
Switzerland) in sterile medium. The final concentration of S9 in
500e3.9 mg/mL doses. After 24 h of incubation, the cytotoxicity test
was performed using the In Cytotox-XTT 1 Parameter Cytotoxicity
Kit (Xenometrix AG, Gewerbertrasse 25, Switzerland), which
measures mitochondrial activity (tetrazolium hydroxide (XTT)) in
NIH/3T3 and A549 cells. Firstly, the cells were washed with phos-
the assay was 4.5% v/v. 2-nitrofluorene (2
mg/mL) and 4-
nitroquinoline N-oxide (0.1 g/mL) were used as positive con-
m
trols in the absence of S9 mix. In the presence of S9 with TA 98
and TA100, 2-aminoanthracene was used as a positive control in
phate buffered saline (PBS) and were added 200
culture medium. XTTI and XTTII solution were mixed at 1:100 ratio.
Then, 50 L of this mixture was added to all wells. The plate was
mL/well of fresh
the concentrations of 1
DMSO was used as a solvent control. Then, each well of a 24-well
plate received 10 L of each test chemical solution. Then, exposure
mg/mL and 2.5
mg/mL, respectively. 4%
m
incubated for 3 h at 37 ꢁC, 5% CO2. After 3 h, the content of the well
was mixed by pipetting up and down. Then, OD of the plate was
read at 480 nm with a reference wave length at 680 nm. Inhibition%
was calculated at each concentration of the compounds. IC50 value
was estimated by non-linear regression analysis. Cisplatin was used
as a positive control. The stock solutions of the compounds were
prepared in DMSO and further dilutions were made with fresh
m
medium (Xenometrix AG, Gewerbertrasse, Switzerland) was
mixed with the bacterial culture at a ratio of 1:10 (TA98) and 1:20
(TA100). For the experiments with S9 mix, the volume of the
exposure medium was reduced accordingly. 240
mL of this mixture
was added to the wells of the 24-well-plate. Then, plates were

352
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.055.
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